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Open AccessVol 8 No 6 Research article Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus Irene EM Bultink1,2

Open Access

Vol 8 No 6

Research article

Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus

Irene EM Bultink1,2,3, Dörte Hamann4, Marc A Seelen5, Margreet H Hart4, Ben AC Dijkmans1,2,3, Mohamed R Daha5 and Alexandre E Voskuyl1

1 Department of Rheumatology, VU University Medical Center, Postbox 7057, Amsterdam 1007 MB, The Netherlands

2 Slotervaart Hospital, Louwesweg 6, Amsterdam 1066 EC, The Netherlands

3 Jan van Breemen Institute, Dr J van Breemenstraat 2, Amsterdam 1056 AB, The Netherlands

4 Sanquin Research at CLB, Plesmanlaan 125, Amsterdam 1066 CX, The Netherlands

5 Department of Nephrology, Leiden University Medical Center, Postbox 9600, Leiden 2300 RC, The Netherlands

Corresponding author: Irene EM Bultink, iem_bultink@hotmail.com

Received: 17 Jun 2006 Revisions requested: 8 Aug 2006 Revisions received: 9 Sep 2006 Accepted: 13 Dec 2006 Published: 13 Dec 2006

Arthritis Research & Therapy 2006, 8:R183 (doi:10.1186/ar2095)

This article is online at: http://arthritis-research.com/content/8/6/R183

© 2006 Bultink et al.; licensee BioMed Central Ltd

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Infection imposes a serious burden on patients with systemic

lupus erythematosus (SLE) The increased infection rate in SLE

patients has been attributed in part to defects of immune

defence Recently, the lectin pathway of complement activation

has also been suggested to play a role in the occurrence of

infections in SLE In previous studies, SLE patients homozygous

for mannose-binding lectin (MBL) variant alleles were at an

increased risk of acquiring serious infections in comparison with

patients who were heterozygous or homozygous for the normal

allele This association suggests a correlation between

functional MBL level and occurrence of infections in SLE

patients We therefore investigated the biological activity of

MBL and its relationship with the occurrence of infections in

patients with SLE Demographic and clinical data were

collected in 103 patients with SLE Functional MBL serum levels

and MBL-induced C4 deposition were measured by

enzyme-linked immunosorbent assay using mannan as coat and an

or C4b-specific monoclonal antibody The complete

MBL-dependent pathway activity was determined by using an assay

that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with

a specific monoclonal antibody against C5b-9 Charts were systematically reviewed to obtain information on documented infections since diagnosis of SLE Major infections were defined

as infections requiring hospital admission and intravenous administration of antibiotics In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented in the 103 SLE patients (mean age 41 ± 13 years, mean disease duration 7 ± 4 years) The percentage of SLE patients with severe MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively Although deposition of C4 to mannan and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels nor MBL pathway activity was associated with infections or major infections in regression analyses In conclusion, SLE patients frequently suffer from infections, but deficiency of functional MBL does not confer additional risk

Introduction

Infections are an important cause of morbidity and mortality in

patients with systemic lupus erythematosus (SLE) Infectious

complications occur in 25% to 45% of SLE patients in case

series [1,2], and infection as cause of death has been reported

in up to 50% of patients with SLE [3,4] The increased

infec-tion rate in patients with SLE has been attributed in part to

defects in the complement system, which has an important

role in host defence against microorganisms [3]

Genetic deficiencies of early components of the classical pathway of complement activation are strongly associated with the development of SLE [5] In particular, deficiency of C1q is a major predisposing risk factor for SLE C1q plays a role in the recognition and clearance of apoptotic material [6] and binds predominantly to antibodies and protein structures

on bacteria and viruses, resulting in complement activation More recently, the lectin pathway of complement activation has also been suggested to play a role in the pathogenesis of HRP = horseradish peroxidase; MASP = mannose-binding lectin-associated serine protease; MBL = mannose-binding lectin; SLE = systemic lupus erythematosus.

SLE [7] and in the occurrence of infections in SLE [8-10].

Mannose-binding lectin (MBL) is a serum protein with

charac-teristics very similar to those of C1q [11] MBL may activate

complement through the lectin pathway by interacting with

MBL-associated serine proteases (MASPs) Furthermore,

MBL can directly opsonise pathogens and enhance the

activ-ity of phagocytes [12]

Homozygosity for variant MBL alleles is probably a minor risk

factor for the presence of SLE, as shown by a recent

meta-analysis of all available case-control studies In that study, a

significant association between MBL codon 54 variant B and

SLE was demonstrated [13] Genetic and phenotypic

defi-ciency in producing MBL has been associated with recurrent

or serious infections, mainly in children [14] and in

immune-compromised individuals [15]

In three previous studies, SLE patients homozygous for MBL

variant alleles were at an increased risk for serious infections

compared with patients who were heterozygous or

homozygous for the normal allele [8-10] This association

sug-gests a correlation between functional MBL level and

occur-rence of infections in these patients However, the association

between rates of infection and functional MBL serum levels

has not been studied yet

Genotype predicts MBL serum concentration reasonably well

at the population level However, in individuals, even full

geno-typic characterisation is insufficient to predict functional MBL

serum levels [16,17] Moreover, functional MBL serum levels

not only are determined by MBL genotype and promoter

poly-morphisms but also are influenced by MASP activity and

serum levels of other complement factors Assays are available

to test these separate influences [18] The capacity of highly

oligomerised MBL to bind to microorganisms can be tested in

vitro by incubating serum on mannan-coated plates and

sub-sequently detecting bound MBL with an MBL-specific

mono-clonal antibody This assay is dependent only on the amount of

functional MBL protein Activity of the MBL/MASP complex is

determined by performing the incubation on mannan-coated

plates at 37°C and subsequently detecting C4 deposition on

the surface Functional MBL serum levels and C4 deposition

are highly correlated except in cases of MASP deficiency

Functional activity of the entire MBL pathway can be measured

starting with binding of MBL to mannan and detection of the

membrane-attack complex C5–9 This assay is sensitive to

defects in all components of the MBL pathway Following the

above methodology, we measured functional MBL activity in

our clinic population of patients with SLE and related this to

past infectious events

Materials and methods

Patients and data collection

One hundred and three patients fulfilling the revised criteria for

the classification of SLE [19] were included in the study All

patients were regular outpatients of the rheumatology clinic of the VU University Medical Center, the Jan van Breemen Insti-tute, or the Slotervaart Hospital These clinics provide primary through tertiary care for patients with SLE in Amsterdam, The Netherlands The local ethics committee approved the study All patients provided informed consent for their participation Demographic, patient, and disease characteristics were sys-tematically recorded by interview, self-reported questionnaire, chart review, and clinical examination performed by one rheu-matologist (IEMB)

Definition of infections

Infections were included in the analysis if they met the follow-ing criteria: documentation in medical records (patient charts and microbiology laboratory reports), occurring since diagno-sis of lupus, and confirmed both by clinical findings and posi-tive cultures If bacterial isolates were not available (for example, in some cases of pneumonia, sinusitis, and otitis), infection was diagnosed by clinical manifestations in combina-tion with radiographic findings and response to antibiotic treat-ment Lower urinary tract infections were excluded because of potential under-reporting of this infection Viral infections were included if clinical symptoms of a viral infection were present

in combination with positive culture or confirmed by high titres

of antibodies for virus-specific antigens Herpes zoster infec-tions were included if typical dermatomal vesicular cutaneous lesions had occurred Major infections were defined as infec-tions for which hospital admission and intravenous antibiotic treatment had been required Opportunistic infections were defined as infections with pathogens that are uncommon in the non-compromised host

Laboratory investigations

Assessment of functional MBL serum levels and C4 concentration

Fifty micrograms per millilitre of mannan (Sigma-Aldrich, St Louis, MO, USA) was coated overnight in coating buffer (0.1

M carbonate, pH 9.6) on a Nunc Maxisorp microtitre plate (Inv-itrogen, Breda, The Netherlands) All incubations were per-formed in a volume of 100 μl at room temperature Plates were washed five times with water (all other washes were also car-ried out in water), and samples were diluted 1:3, 1:9, 1:27, and 1:81 in TTG/Ca2+ (20 mM Tris, 150 mM NaCl, 0.2% gel-atin [wt/vol], 0.02% TWEEN-20 [wt/vol], 10 mM CaCl2, pH 7.4) for 1 hour A standard curve was generated using two-fold serial dilutions of a pool of 3,000 sera obtained from blood bank donors, containing 1.5 μg/ml of MBL when compared with a standard from Dade Behring Holding GmbH (Eschborn, Germany) Plates were washed five times and incubated with

2 μg/ml of biotinylated CLB-MBL/1 in TTG/Ca2+ for 1 hour After washing five times, the plates were incubated with polymerised streptavidin-horseradish peroxidase (HRP) (San-quin, Amsterdam, The Netherlands) diluted 1:10,000 in Tris-buffered saline/Ca2+/milk (20 mM Tris, 150 mM NaCl, 10 mM CaCl2, 2% [vol/vol] cow's milk, pH 7.4) Plates were incubated

for 30 minutes and washed five times The assay was

devel-oped with 100 μg/ml TMB (3,3',5,5'-tetramethylbenzidine) in

0.11 M sodium acetate (pH 5.5) containing 0.003% H2O2

(vol/vol) Substrate conversion was stopped by adding 100 μl

of 2 M H2SO4, and absorbance was measured at 450 nm The

assay was validated by testing 100 blood bank donors using

either CLB-MBL/1 as described above or HYB 131-1 (a kind

gift of S Thiel, University of Aarhus, Aarhus, Denmark), which

revealed comparable results A cutoff value of less than 0.05

μg/ml MBL corresponds to low-level-producing MBL

geno-types [16] Serum levels of C4 were measured by

nephelom-etry (Dade Behring Holding GmbH)

Assessment of MBL-induced C4 deposition on mannan

Plates were coated overnight in coating buffer with 50 μg/ml

of mannan All incubations were performed in a volume of 100

μl Serum samples were diluted 1:10, 1:30, 1:90, and 1:270

in VB/T (10 mM veronal, 150 mM NaCl, 10 mM CaCl2, 10 mM

MgCl2, 0.3% bovine serum albumin [wt/vol], 0.02%

TWEEN-20) and incubated for 30 minutes at 37°C A standard curve

was generated using two-fold serial dilutions of a pool of

3,000 sera obtained from blood bank donors, containing 1.5

μg/ml of MBL Plates were washed five times with water (all

other washes were also carried out in water) and incubated for

1 hour with biotinylated monoclonal antibody C4–10 [20] for

measuring C4b deposition, at 0.25 μg/ml in TTG/Ca2+ Plates

were washed five times and incubated with polymerised

streptavidin-HRP as described above Plates were washed

five times and colour reaction was obtained and measured as

described above Individual results are expressed as

percent-age of the standard pool serum, which was set at 100%

Val-ues below 10% point to severely decreased activity In healthy

controls, 90% of individuals with a C4 deposition activity

below 10% have a functional MBL serum level of less than 0.1

μg/ml

Alternative pathway activation is excluded by diluting the

serum at least 1:10 Classical pathway activation does not

influence the assay, because addition of monoclonal antibody

C1q-85 [21], which inhibits activation of C1q by immune

com-plexes, has no effect on the results In addition, use of

MBL-deficient serum has never resulted in detectable levels of C4

deposition under the described conditions

C4 deposition assay results are not influenced by a low C4

concentration in serum that can be expected in patients with

SLE Serum of two healthy donors was tested in a dilution

curve in the presence of a constant dilution of 200 ng/ml

nat-ural purified human MBL (a kind gift from Inga Laursen,

Stat-ens Serum Institute, Copenhagen, Denmark) C4 deposition is

reduced at C4 values below 0.001 g/l Because sera with low

C4 deposition (<10%) are evaluated at a 1:10 dilution, the

assay can be performed until a minimal C4 concentration of

0.01 g/l (Figure 1)

Antibodies

The monoclonal antibody to C4b (anti-C4–10) was generated

by fusing spleen cells from mice immunised with native C4 with the mouse myeloma cell line SP2/0 [20] CLB-MBL/1 was obtained by a fusion of spleen cells from a mouse immu-nised with natural MBL, purified from Cohn fraction III (San-quin) as previously described by Kilpatrick [22] Specificity for MBL was shown on Western blot Affinity for the carbohy-drate-binding domain was concluded from its ability to inhibit binding of MBL to mannan (data not shown)

Assessment of MBL pathway activation

MBL pathway activation was assessed by an enzyme-linked immunosorbent assay technique that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with a specific monoclonal anti-body against C5b-9, as described previously [23] To prevent contribution of the classical pathway in this assay, an antibody against C1q is added to the reaction mixture [23] Functional activity of the MBL pathway was expressed as percentage of

a standard consisting of pooled human serum set at 100% Decreased functional activity of the MBL pathway was defined

as functional activity of less than 10%

Statistical analyses

Differences between groups were evaluated by Mann-Whit-ney test; the following subgroups were considered: SLE patients with one or more major infections versus SLE patients without major infections, SLE patients with one or more major infections versus patients who experienced minor infections only, and SLE patients with one or more major infections ver-sus SLE patients without any infections Correlations between

Figure 1

Dependence of C4 deposition assay on C4 serum concentration

Dependence of C4 deposition assay on C4 serum concentration C4 deposition was measured in a dilution curve of serum of two healthy donors in the presence of a constant mannose-binding lectin concen-tration of 200 ng/ml C4 deposition becomes C4-dependent at a con-centration below 0.001 g/l Because sera with low C4 deposition (<10%) are evaluated at a dilution of 1:10, the minimal required C4 concentration in a tested serum will be 0.01 g/l.

different assays and between assays and disease activity

scores were evaluated by calculating Spearman's correlation

coefficient Variables possibly associated with the occurrence

of infections and major infections were first examined by

uni-variate tests and subsequently by multiple regression analysis

As a consequence of the low number of opportunistic

infec-tions since lupus onset in our patients, a further analysis of

var-iables associated with the occurrence of opportunistic

infections was not performed The following variables were

evaluated in relationship to the occurrence of the first major

infection since lupus diagnosis by univariate analyses: disease

duration, history of lupus nephritis confirmed by renal biopsy

occurring before the first major infection, functional MBL

serum levels, activity of the MBL pathway of complement

acti-vation, use of oral corticosteroids at the moment of first major

infection, and previous use within the last 3 months before the

first major infection of the following drugs: intravenous

methyl-prednisolone, hydroxychloroquine, methotrexate, azathioprine,

and (oral and/or intravenous) cyclophosphamide To

deter-mine which factors were significantly associated with

infec-tions and major infecinfec-tions, the demographic, clinical, and

therapy variables with a p value of less than 0.2 in the

univari-ate analyses and variables with supposed clinical relevance

were entered into the respective multiple regression analyses

The multiple regression models were refined by tentatively

adding to the (almost) final model single variables initially not

included in the model, so as to check once more whether

these variables could indeed be missed A two-sided p value

of less than or equal to 0.05 was considered statistically

sig-nificant The software used was the Statistical Package for

Social Sciences for Windows, version 13.0 (SPSS Inc.,

Chi-cago, IL, USA)

Results

Patient characteristics

The demographic, clinical, and therapy characteristics of the

103 patients with SLE are shown in Table 1 The majority of

the patients were female Caucasians with a mean disease

duration of ± 7 years The ethnic backgrounds of the

remain-ing 23% of patients were Asiatic (11%), negroid (8%),

Medi-terranean (3%), and other (1%) The disease activity and

organ damage index were modest in most patients at the time

of inclusion Most patients had been treated with

corticoster-oids and hydroxychloroquine in the past At the moment of

inclusion, half of the patients were on treatment with both

cor-ticosteroids and hydroxychloroquine and 16% were treated

with azathioprine

Functional MBL serum levels

The median functional MBL serum level in the patients with

SLE (1.4 μg/ml, range 0.04 to 7.60 μg/ml) was comparable

with that in 100 healthy laboratory workers (1.1 μg/ml, range

0.02 to 11.2 μg/ml) The prevalence of severely decreased

MBL levels (<0.05 μg/ml) was similar to that of healthy

labora-tory workers (13% and 14%, respectively)

Complement C4 deposition and MBL pathway activity

As shown in Table 1, the median C4 deposition in patients with SLE was 71% versus 114% in the healthy controls and was less than 10% of the activity of the standard in 21% of the patients with SLE versus 16% in healthy controls The median functional activity of the MBL pathway in patients with SLE was 16% versus 56% in 120 healthy controls, and functional activity of the MBL pathway was less than 10% of the activity

of the standard in 43% of the patients with SLE versus 28%

in healthy controls (Table 1)

Correlations between functional MBL level, biological activity of MBL, complement C4 deposition, and disease activity in patients with SLE

Both MBL pathway activation and C4 deposition were

corre-lated to MBL serum levels (r = 0.75 and r = 0.66, respectively).

In addition, C4 deposition was correlated to MBL pathway

activity (r = 0.77, all correlations p = 0.0001 or less) C4 levels

were poorly correlated to MBL pathway activity and C4

depo-sition (r = 0.2, p = 0.047 and r = 0.26, p = 0.009,

respec-tively) MBL pathway activity was poorly correlated to C3 level

(r = 0.2, p = 0.046) No consistent associations between

func-tional MBL activity, measured by the three assays, and SLEDAI (systemic lupus erythematosus disease activity index) or ECLAM (European consensus lupus activity measurement) disease activity scores were found (data not shown)

Infectious episodes

Fifty-one patients with SLE (50%) had suffered at least one infectious episode since lupus onset The number of infections

in a single patient ranged from zero to nine, and the mean (± standard deviation) number of infections was 1.1 (± 1.7) A total of 115 infectious episodes were documented, of which 37% were major infections As shown in Table 2, the most common locations of infections were skin and mucosa (29%), lower respiratory tract (22%), upper respiratory tract (14%), genital (11%), gastrointestinal tract (9%), and systemic (7%) The most common infection was Herpes zoster skin infection (16%) Microorganisms were isolated in 50% of the infectious episodes: bacteria (45%), viruses (38%), and yeasts (12%) Twenty-three patients had a total of 42 major infections These were most commonly located in the lower respiratory tract (31%), systemic (21%), genital tract (10%), and skin and mucosa (10%) In cases in which the causal microorganism could be identified (57%), 71% proved bacterial, 17% viral, 4% fungal, and 4% a combination of viral and fungal

infec-tions Staphylococcus aureus was the most frequent isolate

(17%) Five patients had an opportunistic infection:

oesopha-geal infection caused by Candida albicans (2), pneumonia due to Klebsiella pneumoniae (1), sinusitis due to Aspergillus

fumigatus (1), and one sepsis caused by Cytomegalovirus and

C albicans.

Table 1

Demographic, clinical, and therapy variables

103)

Healthy controls

Demographic variables

Comorbidity

Clinical and laboratory variables

MBL-induced C4 deposition on mannan, median (range) percentage 71 (1.1–399) 114 (3–494)

Functional activity of the MBL pathway, median (range) percentage 16 (0.1–119) 56 (0–133)

Therapy variables

Oral corticosteroids

Except where indicated otherwise, values are presented as the mean ± standard deviation ECLAM, European consensus lupus activity

measurement (range 0 to 10) [35]; MBL, mannose-binding lectin; SLEDAI, systemic lupus erythematosus disease activity index (range 0 to 105) [36]; SLICC/ACR, Systemic Lupus International Collaborating Clinics/American College of Rheumatology [37].

Variables associated with major infectious episodes

Univariate analyses

Results of univariate analyses of potential risk factors for major

infections are shown in Table 3 Functional MBL serum levels

and functional activity of the MBL pathway of complement

acti-vation were not different between SLE patients with major

infections and those without major infections As expected,

SLE patients who had suffered at least one major infection

since lupus diagnosis had a significantly longer mean (±

standard deviation) disease duration in comparison with SLE

patients who never had a major infection Furthermore, the

per-centage of SLE patients with a previous SLE

glomerulonephri-tis was significantly higher in SLE patients who had major

infections than in SLE patients without major infections The

median serum creatinine level in SLE patients at the moment

of the first major infection (74 μmol/l, range 45 to 389 μmol/l)

was comparable with that in SLE patients without major

infec-tions (82 μmol/l, range 65 to 154 μmol/l) Prevalence of

hydroxychloroquine use within the last 3 months before the

first major infection was significantly lower than the prevalence

of hydroxychloroquine use ever in SLE patients without a major

infection (p = 0.0001).

Multiple regression analyses

As shown in Table 4, disease duration was significantly

posi-tively associated and hydroxychloroquine use was significantly

negatively associated with the occurrence of the first major

infection in a multiple regression analysis that included the

fol-lowing variables: disease duration at follow-up; previous lupus

nephritis; use of hydroxychloroquine, intravenous

methylpred-nisolone, and intravenous cyclophosphamide within the last 3

months before the first major infection; use of each of these

drugs ever in case no major infection occurred (as independ-ent variables); and first major infection (as dependindepend-ent varia-ble) None of the other variables investigated demonstrated a significant contribution to this model

Discussion

Our patients with SLE frequently suffered from major infec-tions, but we found no association between functional activity

of the MBL pathway and the occurrence of infection Strengths of our study include a high number of clinically rele-vant events and to our knowledge the first attempt to study functional MBL in SLE patients at risk for infection

Functional activity of the MBL pathway in serum not only is determined by mutations in the gene encoding MBL, but also

is influenced by promoter polymorphisms Moreover, biologi-cal activity of MBL also depends on MASP activity and envi-ronmental factors can influence MBL levels in serum [16,24] MBL is a weak acute-phase reactant [12,25], and circulating MBL levels were found to increase only 1.5- to 3-fold in non-SLE patients during an acute-phase reaction [25] No increase was observed in individuals who were homozygous

or compound heterozygous for MBL mutant alleles [26], and therefore quite stable MBL levels are present in individuals For these reasons, functional MBL activity is thought to be a

better estimate of the in vivo situation than nucleic acid

substi-tutions determining genotypes when evaluating the role of the MBL pathway of complement activation in relation to the occurrence of infections in SLE

In the present study, functional MBL activity was measured by three assays None of these assays showed an association

Table 2

Anatomic site of infections (n = 115) and isolated microorganisms in 103 patients with systemic lupus erythematosus

Anatomic site of infections (total number) Microorganism

Skin and mucosa (33) Herpes zoster (18), Staphylococcus aureus (1), Candida albicans (2),

unidentified (12) Lower respiratory tract (25) S aureus (2), Klebsiella pneumoniae (1), Haemophilus influenzae (1),

Respiratory syncytial virus (1), unidentified (20) Upper respiratory tract (16) Herpes simplex (2), Aspergillus fumigatus (1), unidentified (13)

Gardnerella vaginalis (1), Human papillomavirus (1), Bacteroides fragilis (1), unidentified (3)

Gastrointestinal tract (10) Salmonella typhi (3), Campylobacter jejuni (2), C albicans (2),

unidentified (3)

albicans (1), unidentified (1)

Musculoskeletal (4) Osteomyelitis by S aureus (1), septic arthritis of knee prosthesis by S

aureus (1), osteomyelitis by unidentified microorganism (2)

cervical abscess), unidentified (post-operative abdominal abscess) Microorganisms were isolated in 57 (50%) of the 115 infections.

between deficient MBL activity and the occurrence of

infec-tions or major infecinfec-tions in patients with SLE The prevalence

(13%) of severe MBL deficiency in SLE patients, defined as

MBL serum levels of less than 0.05 μg/ml, was similar to that

in healthy laboratory workers Functional MBL level, biological

activity of MBL, and complement C4 deposition assays were significantly correlated However, the prevalence of decreased C4 deposition (21%) and decreased MBL pathway activity in 43% of the patients with SLE was higher than the prevalence

of decreased MBL serum levels in patients with SLE As

Table 3

Potential risk factors for the first major infection in 103 patients with SLE

= 1 major infection (n = 23) No major infection (n = 80) p value

Laboratory variables

Functional MBL serum level, μg/

Functional MBL serum level <0.05

MBL-induced C4 deposition on

mannan, percentage a

MBL-induced C4 deposition on

mannan <10%

MBL pathway activity,

Clinical variables

Disease duration at follow-up,

years

Treatment variables Use <3 months before the first

major infection

Previous use ever since lupus

diagnosis

Except where indicated otherwise, values are presented as the mean ± standard deviation a Values are presented as the median (range) MBL, mannose-binding lectin; SLE, systemic lupus erythematosus.

Table 4

Multivariate analysis of the first major infection (dependent variable) and clinical and therapy variables (independent variables)

a Use within the last 3 months before the first major infection or previous use since lupus diagnosis if no major infection SLE, systemic lupus erythematosus.

expected, both C4 and C3 levels were lower in SLE patients

than in healthy controls However, correlations between C4 or

C3 and C4 deposition and MBL pathway activity were very

low MBL-induced complement activity was correlated to MBL

levels in patients and healthy controls Our findings are in

con-trast to a previous study in which MBL-induced C4 deposition

was associated with plasma C4 levels in healthy controls and

patients with SLE [27] In that study, MBL levels were

corre-lated to C4 deposition in healthy controls but not in patients

with SLE, suggesting a high sensitivity of that assay for C4

lev-els Our C4 deposition assay is dependent primarily on

func-tional MBL plasma levels in both healthy controls and patients

with SLE, and C4 is not a limiting factor until very low

concen-trations of 0.01 g/l in healthy controls Although all patients

with SLE had levels above the threshold, dysfunction of C4 or

the presence of complement inhibitors in SLE plasma cannot

be excluded, because MBL-induced complement activity is

reduced in our patients Furthermore, additional consumption

of complement factors other than C4 may be relevant because

we observed a higher frequency of deficient patients in the

MBL pathway activity assay compared with the C4 deposition

assay Further studies are needed to investigate the role of the

several complement deficiencies, dysfunction, inhibitors, and

consumption

To exclude an influence of very low C4 serum levels on both

C4 deposition and MBL pathway activity assays, subanalyses

of the association between laboratory variables and the

occur-rence of infections and major infections were performed after

exclusion of SLE patients with C4 serum levels of less than or

equal to 0.1 g/l (n = 28) In these subanalyses, C4 deposition

assay and MBL pathway activity assay were not associated

with infections or major infections, either in univariate and

mul-tiple regression analyses (data not shown)

When functional MBL activity with respect to the severity of

infections was analysed, no significant differences were found

between median values of functional MBL serum level, C4

deposition, and MBL pathway activity in three subgroups of

patients: SLE patients with one or more major infections,

patients with SLE who experienced minor infections only, and

SLE patients without any infections (Figure 2)

Associations between MBL genotype and the occurrence of

infections in patients with SLE have been reported [8-10] In

those studies, MBL serum levels were measured as well, but

no association between low MBL serum levels and the

occur-rence of infections was demonstrated, probably because of

the inability of serologic methods used to distinguish between

functional and nonfunctional protein [28] Unfortunately, no

DNA is available from our patient cohort and for this reason we

cannot correlate MBL genotype and risk of infections As far

as data are available, differences in clinical and

epidemiologi-cal characteristics of the study patients are unlikely to be

responsible for the discrepancy between the results of the

Figure 2

Mannose-binding lectin (MBL) serum level, C4 deposition, and MBL pathway activity

Mannose-binding lectin (MBL) serum level, C4 deposition, and MBL pathway activity Functional MBL activity measured by three assays in

all patients with systemic lupus erythematosus (SLE; n = 103), a sub-group of SLE patients without infections (SLEno; n = 52), a subsub-group

of patients with SLE who had minor infections only (SLEmin; n = 28), a subgroup of SLE patients with one or more major infections (SLEmaj; n

= 23), and healthy controls (a) MBL serum levels (b) Complement C4 deposition (c) MBL pathway activity Each datapoint represents one

patient Bars show the median values No significant differences were found between the median functional MBL activity of all patients with SLE and each of the subgroups or between the subgroups, as meas-ured with three assays (data not shown).

present study and previous studies [8-10], except for the

dif-ferent racial background of the study patients in the Japanese

study [10] Another explanation for the discrepancy between

the genotypic and the phenotypic data could be that

unidenti-fied linkages between mutations or polymorphisms in the MBL

gene with mutations or polymorphisms in other genes might

influence the genetic approach

Despite the use of new treatment strategies to improve the

clinical outcome in patients with SLE, the importance of

infections as a cause of morbidity and mortality in SLE has not

changed in the past decades Defects of immune defence and

treatment with corticosteroids and immunosuppressive agents

are supposed to play a role in the pathogenesis of infections

in SLE [3], but the mechanisms underlying the increased

infec-tion rate in SLE are not fully understood Our study

demon-strates the occurrence of at least one infectious episode since

lupus diagnosis in 50% of patients with SLE, and this high

infection rate is confirmed by other studies reporting infectious

complications in up to 45% of SLE patients in case series

[1,29] The spectrum of infections found in our study is in line

with other studies in patients with SLE, which report a broad

spectrum of infections caused predominantly by

community-acquired bacteria [1,29,30] The severity of infections found in

other studies in patients with SLE [1,30] is confirmed by our

study, which shows a third of the infections in patients with

SLE to be major infections for which hospital admission was

required Furthermore, the high incidence of Herpes zoster

infection, occurring at least one time in 14% of the patients, is

in line with other studies in patients with SLE [1,31]

With respect to major infections, use of immunosuppressive

medication and use of corticosteroids were not associated

with the occurrence of the first major infection in our patients

in the best multiple logistic regression model This finding is in

line with a previous study on major infections in SLE patients

in the Hopkins Lupus Cohort [32]

Renal insufficiency is a possible risk factor for infections

How-ever, no such association was found in our study The

presence of prior SLE glomerulonephritis was not significantly

associated with the occurrence of the first major infection in

the best multiple logistic regression model Moreover, the

median serum creatinine level at the moment of the first major

infection was similar to that in SLE patients without major

infection

Our study demonstrated a significant negative association

between hydroxychloroquine use and the occurrence of major

infections This finding might be explained by the predominant

use of hydroxychloroquine in the treatment of patients with

mild lupus disease activity and not by the antimicrobial

proper-ties of hydroxychloroquine Antimalarials act against

patho-genic organisms that are very uncommon in Western Europe

Limitations of the present study are the racial and socioeco-nomic backgrounds of the study population As a conse-quence of the rather high percentage of Caucasians in the study population, the associations found in the present study may not be generalised to lupus cohorts of different racial background However, the disease severity in our study popu-lation appears to be comparable to that in other large multi-ethnic SLE cohorts with respect to current corticosteroid use [33,34], prevalence of renal disease [1,30,33,34], mean organ damage index [1,34], and mean disease activity score [30,33], suggesting that race may not have had an important influence

on our conclusions Socioeconomic status, a factor influenc-ing disease severity and prognosis in patients with SLE, was not assessed in the present study Therefore, the results of our study may not be generalised to SLE cohorts of different soci-oeconomic background

Conclusion

The results of the present study emphasise that infection imposes a serious burden on patients with SLE Although defects in the complement system have been suggested to be partially responsible for the high infection rate in patients with SLE, the results of our study suggest that deficiency of func-tional MBL activity does not play a role in the susceptibility to infections or major infections

Competing interests

The authors declare that they have no competing interests

Authors' contributions

IEMB collected clinical data, carried out the statistical analy-ses, and drafted the manuscript DH supervised the assess-ment of functional MBL serum levels, C4 concentration, and MBL-induced C4 deposition and helped to draft the manu-script MAS performed the MBL pathway activity enzyme-linked immunosorbent assay (ELISA) and helped to draft the manuscript MHH performed the assessment of functional MBL serum levels, C4 concentration, and MBL-induced C4 deposition BACD participated in the design of the study and reviewed the draft of the manuscript MRD participated in the design of the study, supervised the performance of the MBL pathway activity ELISA, and reviewed the draft of the manu-script AEV participated in the design of the study, contributed

to the coordination of the study, supervised the statistical anal-yses, and reviewed the draft of the manuscript All authors have read and approved the final manuscript

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